Whole exome sequencing identifies novel pathogenic variants in TGM1 and ALOX12B in patients with hereditary ichthyosis.

BACKGROUND: Hereditary ichthyosis is a clinically and genetically heterogeneous disorder associated with more than 50 genes with TGM1, ALOX12B, and ALOXE3 being the most prevalent. Establishing an accurate diagnosis is important for effective genetic counseling and optimal patient management. OBJECTIVE: We studied the diagnostic value of whole exome sequencing (WES) in a small case series with hereditary ichthyosis. METHODS: During a 1-year period, index cases of 5 unrelated families clinically diagnosed with hereditary ichthyosis went through WES, followed by extensive segregation analysis. Prenatal diagnosis (PND) was conducted where indicated. RESULTS: We identified 4 homozygous variants-2 in TGM1 (c.655A > G and c.797A > G) and 2 in ALOX12B (c.527 + 2 T > G and c.1654G > T)-alongside a heterozygous variant in TGM1 (c.428G > A) in 5 families. The variants were all pathogenic/likely pathogenic according to the ACMG classification and segregation analysis, except for c.797A > G in TGM1 which remained a variant of unknown clinical significance. Four variants were novel. All families were referred either during pregnancy or before reproductive planning; 4 benefited from WES as it identified the mutation in the probands and enabled carrier detection in at-risk relatives; PND was conducted in 2 families. CONCLUSION: Our findings further support WES is a powerful tool for the comprehensive, accurate, and rapid molecular diagnosis of hereditary ichthyosis and can offer opportunities for reproductive planning, carrier screening and prenatal diagnosis to at-risk families.

Histological Evaluation of Products of Conception, Who Benefits from It?

Background: Histopathological evaluation of the first trimester pregnancy loss has always been controversial. Although it is recommended, it is not a part of guidelines.Methods: Six hundred eighty-six samples in a referral infertility clinic were evaluated microscopically and categorized. Two hundred ninety-five cases were evaluated by genetic methods (Multiplex Ligation-dependent Probe Amplification).Results: From 569 samples with chorionic villi, 361 cases had history of three or more abortions. 18.3% of this group showed chronic intervillous of unknown etiology (CIUE) and 8.3% revealed intervilli fibrin deposition, both pathologies with a high risk of recurrence. History of a live child was significantly higher in CIUE group. 29% of genetically evaluated cases had a chromosomal abnormality.Conclusion: Histological evaluation of recurrent pregnancy loss could illuminate the cause of abortion in relatively acceptable percentage of cases, especially in mothers with higher number of previous abortion, mothers with a history of live child and in referral centers.

Identification of a novel de novo variant in OTX2 in a patient with congenital microphthalmia using targeted next-generation sequencing followed by prenatal diagnosis.

BACKGROUND: Next-generation sequencing has been proven to be a reliable method for the detection of genetic causes in heterogeneous ocular disorders. In this report an NGS-based diagnostic approach was taken to uncover the genetic etiology in a patient with coloboma and microphthalmia, a highly heterogeneous disease with intrafamilial phenotypic variability. MATERIALS AND METHODS: Next generation sequencing using a targeted panel of 316 genes, was carried out in the proband. Prioritized variants were then identified and confirmed using Sanger sequencing. Prenatal diagnosis of the detected variant was then performed in the family. RESULTS: A novel de novo frameshift variant c.157_164delTTCACTCG (p.Phe53fs) in OTX2, leading to a truncated protein, was identified. Prenatal diagnosis identified the same variant in the fetus. CONCLUSIONS: This report demonstrates the importance of genetic counseling and underscores the efficiency and effectiveness of targeted NGS as a means of detecting variants in inherited eye disorders.

Detection of Aneuploidies in Products of Conception and Neonatal Deaths in Iranian Patients Using the Multiplex Ligation-Dependent Probe Amplification (MLPA).

BACKGROUND: Around 70% of all pregnancies (Including 15% of clinically-recognized ones) are lost due to various fetal or maternal disorders. Chromosomal aneuploidies are among the most common causes of pregnancy loss. Standard chromosome analysis using G-banding technique (Karyotype) is the technique of choice in studying such abnormalities; however, this technique is time-consuming and sensitive, and limited by vulnerabilities such as cell culture failure. The use of molecular cytogenetic techniques, including array-based techniques and Multiplex Ligation-Dependent Probe Amplification (MLPA), has been proposed to overcome the limitations of this method to study the products of conception. This study has been designed to investigate the feasibility of using MLPA technique as a standalone genetic testing, with histopathologic examinations and genetic counseling to detect aneuploidies in products of conception and neonatal deaths. METHODS: Forty-two verified fetal and neonatal samples were studies and genetic counseling was scheduled for all parents. Histopathologic examinations were carried out on the products of conception, and appropriate fetal tissues were separated for genetic studies. Following DNA extraction and purification, MLPA was carried out to investigate chromosomal aneuploidies. RESULTS: Nine samples (21.42%) were diagnosed to be affected with aneuploidy. Detected aneuploidies were trisomy 22 (n=3), trisomy 21(n=1), trisomy 18 (n=2), trisomy 16 (n=1), trisomy 13 (n=1), and monosomy of chromosome X (n=1). The MLPA analysis results were conclusive for all of the fetal samples (Success rate: 100%). CONCLUSION: These results suggest that MLPA, as a standalone genetic testing, is an accurate, rapid, and reliable method in overcoming the limitations of standard cytogenetic techniques in genetic investigation of products of conception.

Non-progressive Nonimmune Hydrops Fetalis Caused by a Novel Mutation in GUSB Gene.

Objectives: Mucopolysaccharidosis type VII (MPS VII) or Sly syndrome is a rare autosomal recessive disorder caused by deficiency of ß-glucuronidase enzyme, which is involved in degradation of glycosaminoglycans. The lack of ß-glucuronidase in this lysosomal storage disorder is characterized by various manifestations such as nonimmune hydrops fetalis, spinal deformity, organomegaly, dysostosis multiplex, intellectual disability, and eye involvement. It is caused by a mutation in GUSB gene located on chromosome 7 q11. The current study reported an Iranian female with MPS VII and a novel mutation (c.542G>T, p.Arg181Leu) in GUSB gene.

Rhodopsin gene mutation analysis in Iranian patients with autosomal dominant retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is the most common hereditary retinal degeneration and an important cause of visual disability worldwide. Rhodopsin gene is one of the most important genes implicated in autosomal dominant RP (ADRP). In this study, we investigated rhodopsin gene mutations in Iranian patients with ADRP. METHODS: Twenty-one patients from 21 unrelated families with a total of 51 affected members were enrolled in this study. After complete history taking, ophthalmic examination and genetic counseling, peripheral blood samples were obtained. Following genomic DNA extraction, all five exons and intron-exon boundaries of RHO gene were sequenced using Sanger method. Interpretation of detected variants was carried out using appropriate databases and bioinformatic tools. Novel variants were screened in 150 unrelated healthy subjects. RESULTS: Results of direct sequencing revealed that five of 21 patients (23.8%) had mutation in the rhodopsin gene. Two of them had previously identified p.P347L mutation, and three had novel variants including p.L95P, p.R177K and p.N310K. None of these novel variants were detected in healthy controls. The p.L95P variant was associated with predominantly inferior retinal involvement. CONCLUSIONS: Our study showed that mutations of the rhodopsin gene are relatively frequent in Iranian patients with ADRP and could be considered in further researches in the future. The novel p.L95P variant may be associated with a specific pattern of retinal degeneration in this population.

Preimplantation High-Resolution HLA Sequencing Using Next Generation Sequencing.

Hematopoietic stem cell transplantation (SCT) is the only therapeutic option in a number of heritable hematologic disorders and hematologic cancers. Many parents and families fail to find an HLA-identical donor for their affected family member. In such cases, conceiving for a "savior baby" remains the only option, especially in countries without access to national registries. By means of next generation sequencing (NGS) techniques, in a single experiment on single-cell products of in vitro fertilization, a healthy HLA-identical embryo can be implanted in the uterus of a concerned mother. The patient can therefore benefit from cord blood SCT along with confirming that the fetuses are not suffering from the heritable disorder. This study is an attempt to study the feasibility of preimplantation HLA sequencing on single blastomeres using NGS. Two couples who had previously undergone preimplantation genetic diagnosis of ß-thalassemia and their overall 10 embryos were studied and their 5 HLA loci were typed in high resolution through multiple displacement amplification and NGS of single cells. For 88.9% of the 90 HLA alleles, conclusive HLA typing in 4 digit sets was made. HLA alleles were typed; 1 ambiguity in the allelic group and 4 ambiguities in the protein level were observed that were then unraveled by haplotype analysis. Amplification efficiency was 93.3% with an allele drop-out (ADO) rate of 22.2% (6 alleles dropped from a maximum of 27 possible ADOs). In this study the feasibility of a new method of preimplantation HLA sequencing via combining the state-of-the-art techniques used in single-cell whole genome amplification, preimplantation genetic diagnosis, and high-resolution HLA typing by NGS has been shown. This method can make preimplantation HLA sequencing a practicable technique in families desperate for an HLA-matched donor.

A novel ACVR1 mutation detected by whole exome sequencing in a family with an unusual skeletal dysplasia.

"Disorganized Development of Skeletal Component" (DDSC) is a group of genetic skeletal dysplasia, caused by mutations in 9 genes including ACVR1. The most known ACVR1-related disorder is fibrodysplasia ossificans progressiva (FOP). FOP variants are frequently encountered with diagnostic challenges due to overlapping clinical manifestations and variable severity. Application of high throughput sequencing methods can overcome these limitations by simultaneous investigation of the entire ACVR1 gene together with other genes involved in disorders with similar manifestations. A 33-year-old man with an unusual skeletal dysplasia and no previous clinical diagnosis is presented in this study. Whole exome sequencing detected a novel c.737T>A (p.Phe246Tyr) mutation in ACVR1 gene. Detailed targeted variant analysis in 226 known genes associated with genetic skeletal disorders together with more specific targeted analysis in 9 genes associated with DDSC ruled out the involvement of other investigated genes. Proband's phenotypically normal father and brother had the same mutation in whom subsequent investigations showed subclinical radiographic findings. The clinical manifestations, the disease course, and the molecular findings of involvement of ACVR1 gene in this family are suggestive of "FOP variant" or an unusual ACVR1-related skeletal dysplasia. Moreover, this report has demonstrated the critical role of the next generation sequencing technique in characterizing such a rare disorder with variable and even no clinical manifestations, providing the opportunity for effective preventive measures such as preimplantation genetic diagnosis.

Prenatal Diagnosis of Tyrosinemia Type 1 Using Next Generation Sequencing.

Prenatal diagnosis using conventional molecular genetic techniques may be encountered with some limitations when the disease causing mutation is unknown. Here, we report on prenatal diagnosis of tyrosinemia in a family with consanguineous marriage and two affected children in whom no disease causing mutation had been identified before pregnancy. Mutation analyses of three genes associated with tyrosinemia including FAH, TAT and HPD were carried out in the fetal DNA sample using Next Generation Sequencing. A heterozygous nonsense mutation (p.Arg237Ter) in FAH gene was detected in the fetus. Further investigations suggested that the fetus was carrier of tyrosinemia type 1. This study demonstrates the successful application of Next Generation Sequencing in prenatal diagnosis, when the time is a limiting factor, more than one (especially large) responsible genes are involved, a "founder" or a "previously detected" mutation is not present and hence the conventional molecular genetic investigations cannot be employed.

Paradox of Modern Pregnancy: A Phenomenological Study of Women's Lived Experiences from Assisted Pregnancy.

The purpose of our study was describing the meaning of pregnancy through Assisted Reproductive Technologies (ARTs). A qualitative design with hermeneutic phenomenology approach was selected to carry out the research. Semistructured in-depth interviews were conducted with 12 women who experienced assisted pregnancy. Three themes emerged from women's experience including finding peace in life, paradoxical feelings, and struggling to realize a dream. We concluded that pregnancy is the beginning of a new and hard struggle for women with fertility problems. The findings of our study resulted in helpful implications for the health care professionals managing assisted pregnancies.

Experiences of Infertile Women Seeking Assisted Pregnancy in Iran: A Qualitative Study.

BACKGROUND: Assisted reproductive technologies (ARTs) are complicated and stressful techniques and the social and cultural norms are major obstacles against their use. Many qualitative studies have been done in the field of women's experiences of infertility, but less is known about the experiences of infertile women seeking assisted pregnancy. The aim of this study was to understand and describe the experience of women who have used assisted reproductive technologies for their current pregnancy. METHODS: This qualitative study was conducted based on a content analysis approach. With purposive sampling, 12 pregnant women who were using ART were recruited from Avicenna Fertility Center in Tehran. Women were selected purposefully and with maximum variation. Interviews were performed after a positive test of pregnancy and women were introduced to researchers in their first visit of pregnancy in the prenatal clinic. Interviews were recorded, transcribed verbatim and analyzed concurrently. Semi-structured interviews were coded, categorized and the themes were also identified. RESULTS: Four main themes were uncovered which included struggle to achieve pregnancy, fear and uncertainty, escape from stigma and the pursuit to achieve husband satisfaction. CONCLUSION: It is essential for these women to be counseled and prepared by their health care providers after the use of ARTs. Distress can be reduced for infertile women seeking assisted pregnancy when they are prepared for possible failures, empowered to deal with stigma, and have their partners' involvement in counseling sessions.

A method for semi-automatic nuchal translucency thickness measurement.

Increased nuchal translucency (NT) thickness is associated with major abnormalities of the heart, great arteries and a wide range of genetic syndromes. NT thickness is one of the most important ultrasound markers in fetal diseases screening. To find an applicable method that accurately detects NT, we use an algorithm based on modified level set for semi-automated segmentation of the region. A regression model is used to find the appropriate direction of NT thickness measurement. The results show 4.5% error.

Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

Correlation of nuchal translucency and thyroxine at 11-13 weeks of gestation.

OBJECTIVE: Nuchal translucency (NT) thickness is one of the major screening markers during the first trimester that could be influenced by several factors. Here, we investigated the association between NT thickness and thyroid related hormones. METHODS: NT thickness was measured with transabdominal ultrasound in 643 pregnant women between 11 and 13 weeks of gestation. Maternal thyroxine (T4), free thyroxine (fT4) and thyroid-stimulating hormone (TSH) were evaluated. Bivariate correlations were assessed and thyroid profile was subcategorized with regard to the calculated reference ranges. RESULTS: An inverse relation was found between serum levels of maternal T4 with NT thickness (r = -0.128, p = 0.001) and CRL (r = -0.168, p < 0.001). TSH and hCG were also found to be correlated (Spearman's correlation coefficient = -0.092, p = 0.019). Prevalence of maternal hypothyroidism and subclinical hypothyroidism were 1.1% and 3.7%, respectively. CONCLUSION: Thyroid function tests are found to independently influence NT measurements in the first trimester. Assessment of hormones such as thyroxine could optimize the interpretation of screening tests for pathological conditions during pregnancy.

Heterozygosity assessment of five STR loci located at 5q13 region for preimplantation genetic diagnosis of spinal muscular atrophy.

Preimplantation genetic diagnosis (PGD) has been considered as an alternative to prenatal diagnosis for prevention of genetic disorders while avoiding the subsequent termination of pregnancy. However, the limited amount of template DNA available in a single diploid cell used for PGD leads to number of problems including an increased incidence of detectable contamination; amplification failure and allele drop out. Due to their highly polymorphic and amplifiable characteristics, short tandem repeat (STR) analysis has been proposed as a mean to overcome these limitations. Heterozygosity of the applied STRs is of paramount importance in their informativity, and should therefore be studied in any certain population. Here, for the first time, we report on the heterozygosity analysis of five STR markers (D5S1408, D5S1417, D5S610, D5S629 and D5S637) flanking to SMA gene region, to examine their applicability in the PGD for SMA disease. We have also investigated other statistical features of these markers and found that all of the five studied STRs were informative and four meet the Hardy-Weinberg equilibrium for the studied population. Furthermore, our results propose that similar approaches can be used for the PGD of other single gene disorders.

Normal ranges for fetal femur and humerus diaphysis length during the second trimester in an Iranian population.

OBJECTIVES: Shortening of the fetal long bones is a sonographic soft marker for screening of Down syndrome in the second trimester that can be influenced by ethnicity. The purpose of this study was to provide normal reference ranges for femur and humerus diaphysis length during the second trimester of pregnancy in an Iranian population. METHODS: This cross-sectional study was performed on 3011 singleton fetuses at 15 to 28 weeks' menstrual age. The relationship between menstrual age and both femur and humerus diaphysis length was determined, and percentile values for each menstrual week were provided. RESULTS: The median femur diaphysis length ranged from 18.05 mm at 15 menstrual weeks to 52.20 mm at 28 menstrual weeks, and the mean humerus diaphysis length ranged from 17.65 mm at 15 menstrual weeks to 48.10 mm at 28 menstrual weeks. There was a linear relationship between menstrual age and both femur diaphysis length (R² = 0.957) and humerus diaphysis length (R² = 0.941). CONCLUSIONS: We have provided normal reference ranges for femur and humerus diaphysis length during the second trimester of pregnancy in an Iranian population.

Familial Williams-Beuren syndrome ascertained by screening rather than targeted diagnosis.

Williams-Beuren syndrome (WBS), a contiguous gene deletion syndrome, mostly occurs sporadically. Although a few cases of familial WBS have been reported in the literature, molecular confirmation of the deletion has not been carried out in all of them. Here, we report on the eighth clinically and molecularly confirmed inherited WBS detected in a family with 'familial mental retardation.' A comprehensive screening approach to mental retardation that included stepwise karyotyping, assessment for fragile-X syndrome, subtelomeric rearrangements and known microdeletion/microduplication syndromes, and a genome-wide array-CGH study was applied. The father, the mother, and their daughter were all mentally handicapped with nonspecific clinical manifestations and dysmorphic features. The first child of the family died from multiple congenital anomalies. The father and his daughter, who had never been suspected to have WBS, were diagnosed as having a deletion of the WBS critical region. No other abnormalities were detected in the family. Unlike other previously reported cases, in which the disease was ascertained on the basis of clinical manifestations, the present report represents an example of the detection of cryptic chromosomal abnormalities in mental retardation patients by the stepwise application of high-throughput screening methods.

Her2 amplification status in Iranian breast cancer patients: comparison of immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH).

INTRODUCTION: Her2/neu is a biomarker which is amplified and/or overexpressed in a subset of breast cancer patients who are eligible to receive trastuzumab. Her-2 gene amplification analysed by fluorescence in situ hybridisation (FISH) and/or protein over-expression detected by immunohistochemistry (IHC) are the two main methods used to detect Her-2 status in clinical practice. The concordance rate between the two techniques is controversial. METHODS: FISH analysis were performed on 104 tumoural samples from breast cancer patients with known IHC results to determine the Her2 gene status. The FISH/IHC analyses results were then compared and the concordance rate was determined. RESULTS: Her2 gene amplification was detected in 0 of IHC score 1+, 24/86 (27.91%) 2+, and 8/13 (61.54%) 3+. The IHC and FISH results concordance rates were 100%, 27.9%, and 61.5% for IHC scores of 1+, 2+, and 3+ respectively. CONCLUSION: The results of this study suggest that IHC 1+ should be considered as negative while IHC 2+ results need further confirmative analysis by FISH. Further quality control and standardization of IHC technique are required to improve the concordance rate between the two methods.

Normal ranges of fetal nasal bone length during the second trimester in an Iranian population.

OBJECTIVE: To provide a normal reference range for nasal bone length (NBL) during the second trimester of pregnancy in an Iranian population. METHODS: This cross-sectional study was performed on 3201 fetuses at 15 to 28 weeks of gestational age (GA). Both singleton and twin fetuses were evaluated. The relationship between NBL and GA was determined and percentile values for each gestational week were provided. RESULTS: NBL measurement was obtained in 98% of singleton and 96% of twin fetuses. There was a linear relationship between GA and NBL both in singleton (R(2) = 0.62) and in twin (R(2) = 0.74) fetuses. There was no significant difference between twins regarding NBL (p = 0.18). CONCLUSION: We have provided the normal reference range for NBL during the second trimester in an Iranian population. NBL in singleton and twin fetuses is similar and there is no significant difference between twins regarding NBL.